Many pathogens produce toxins which are detrimental, and in some cases, lethal, to the host organism. Toxins produced by pathogens can be secreted, or excreted from pathogenic organisms (e.g., xe2x80x9cexotoxinsxe2x80x9d) or toxic structural elements of pathogenic organisms (e.g., xe2x80x9cendotoxins,xe2x80x9d or toxin structural proteins).
Exotoxins are generally proteins or polypeptides. These toxins, which are secreted by the pathogen, can travel within the host and cause damage in regions of the host far removed from the infection site. Symptoms associated with exotoxins vary greatly and include hemolysis, systemic shock, destruction of leukocytes, vomiting, paralysis and diarrhea.
Enterotoxins are exotoxins which act on the small intestine and cause massive secretion of fluid into the intestinal lumen, leading to diarrhea. Enterotoxins are produced by a variety of bacteria and viruses, including the food-poisoning organisms Staphylococcus aureus, Clostridium perfringens, and Bacillus cereus, and the intestinal pathogens Vibrio cholerae, Escherichia coli, and Salmonella enteritidis. 
Endotoxins are lipopolysaccharides/lipoproteins found in the outer layer of the cell walls of gram-negative bacteria. These lipopolysaccharides are bound to the cell membrane and are released upon cytolysis. Symptoms associated with the release of endotoxins include fever, diarrhea and vomiting. Specifically, endotoxins stimulate host cells to release proteins, endogenous pyrogens, which affect the area of the brain which regulates body temperature. In addition to fever, diarrhea and vomiting, the host animal may experience a rapid decrease in lymphocyte, leukocyte, and platelet numbers, and enter into a general inflammatory state.
Although endotoxins are less toxic than exotoxins, large doses of endotoxins can cause death, generally through hemorrhagic shock and tissue necrosis. Examples of bacteria which produce endotoxins include the genera Escherichia, Shigella, and especially Salmonella.
In some cases, the active disease caused by an exotoxin can be treated by administering an antitoxin to the patient. An antitoxin comprises antibodies to the toxin derived from the serum of an animal, typically a horse, which has been immunized by injection of a toxoid, a nontoxic derivative of the toxin. However, the effectiveness of antitoxins is limited because toxins are rapidly taken up by cells and become unavailable to the antibodies. Furthermore, the patient""s immune system can respond to foreign proteins present in the antitoxin, creating a condition known as serum sickness.
Therefore, a need exists for an improved method of treating toxins which significantly reduces or eliminates the above-mentioned problems.
One aspect of the present invention is a method for inhibiting a pathogenic toxin in a mammal, comprising administering to the mammal a therapeutically effective amount of a polymer having a cationic group, such as an amino group, an ammonium group or a phosphonium group, which is connected to the polymer backbone.
The polymer to be administered can be a homopolymer or a copolymer. In one embodiment, the polymer further includes a monomer comprising a hydrophobic group, such as an aryl group or a normal or branched C2-C24-alkyl group.
The polymer to be administered can, optionally, further include a monomer comprising a neutral hydrophilic group, such as a hydroxyl group or an amide group.
Another aspect of the invention is a method for inhibiting a pathogenic toxin in a mammal, such as a human, comprising administering to the mammal a therapeutically effective amount of a polymer comprising a polymethylene backbone which is interrupted at one or more points by a cationic group, such as an amino group, an ammonium group or a phosphonium group.
The present method has several advantages. For example, the polymers employed are easily prepared using standard techniques of polymer synthesis and inexpensive starting materials. The polymers will not be substantially degraded in the digestive tract and, therefore, can be administered orally. Polymer compositions can also be readily varied, to optimize properties such as solubility or water swellability and antitoxin activity.
A description of preferred embodiments of the invention follows.
The present invention relates to a method for inhibiting a pathogenic toxin in a mammal, such as a human, by administering to the mammal a therapeutically effective amount of a polymer comprising a plurality of amino or ammonium groups.
As used herein, the inhibition of a pathogenic toxin refers to the reduction in activity of a toxin produced by a pathogenic microbe. The activity of the toxin can be reduced, for example, by interfering with the production or secretion of the toxin or by binding the toxin to form an inactive complex. Without being bound by theory, one mechanism by which the polymers disclosed herein may inhibit a pathogenic toxin is by binding the toxin.
A xe2x80x9ctherapeutically effective amountxe2x80x9d is an amount sufficient to inhibit, partially or totally, the activity of a pathogenic toxin. The term xe2x80x9cpolymerxe2x80x9d refers to a macromolecule comprising a plurality of repeat units or monomers. The term includes homopolymers, which are formed from a single type of monomer, and copolymers, which are formed of two or more different monomers. A xe2x80x9cterpolymerxe2x80x9d is a copolymer formed from three different monomers. The term polymer, as used herein, is intended to exclude proteins, peptides, polypeptides and proteinaceous materials.
As used herein, the term xe2x80x9cpolymer backbonexe2x80x9d or xe2x80x9cbackbonexe2x80x9d refers to that portion of the polymer which is a continuous chain, comprising the bonds which are formed between monomers upon polymerization. The composition of the polymer backbone can be described in terms of the identity of the monomers from which it is formed, without regard to the composition of branches, or side chains, off of the polymer backbone. Thus, poly(acrylamide) is said to have a poly(ethylene) backbone substituted with carboxamide (xe2x80x94C(O)NH2) groups as side chains.
The term xe2x80x9cpolymer side chainxe2x80x9d or xe2x80x9cside chainxe2x80x9d refers to the portion of a monomer which, following polymerization, forms a branch off of the polymer backbone. In a homopolymer, all of the polymer side chains are identical. A copolymer can comprise two or more distinct side chains. When a side chain comprises an ionic unit, for example, the ionic unit depends from, or is a substituent of, the polymer backbone, and is referred to as a xe2x80x9cpendant ionic unitxe2x80x9d. The term xe2x80x9cspacer groupxe2x80x9d, as used herein, refers to a polyvalent molecular fragment which is a component of a polymer side chain and connects a pendant moiety to the polymer backbone. The term xe2x80x9caliphatic spacer groupxe2x80x9d refers to a spacer group which does not include an aromatic unit, such as a phenylene unit.
The term xe2x80x9caddition polymerxe2x80x9d, as used herein, is a polymer formed by the addition of monomers without the consequent release of a small molecule. A common type of addition polymer is formed by polymerizing olefinic monomers, wherein monomers are joined by the formation of a carbon-carbon bonds between monomers, without the loss of any atoms which compose the unreacted monomers.
The term xe2x80x9cmonomerxe2x80x9d, as used herein, refers to both (a) a single molecule comprising one or more polymerizable functional groups prior to or following polymerization, and (b) a repeat unit of a polymer. An unpolymerized monomer capable of addition polymerization, can, for example, comprise an olefinic bond which is lost upon polymerization.
The term xe2x80x9ccationic groupxe2x80x9d, as used herein, refers to a functional group which bears a net positive charge or a basic group which gains a net positive charge upon protonation at physiological pH. Suitable cationic groups include ammonium groups, such as primary, secondary, tertiary and quaternary ammonium groups; amino groups, such as primary, secondary and tertiary amino groups; sulfonium groups; and phosphonium groups.
The quantity of a given polymer to be administered will be determined on an individual basis and will be determined, at least in part, by consideration of the individual""s size, the severity of symptoms to be treated and the result sought. The polymer can be administered alone or in a pharmaceutical composition comprising the polymer, an acceptable carrier or diluent and, optionally, one or more additional drugs.
The polymers can be administered, for example, topically, orally, intranasally, or rectally. The form in which the agent is administered, for example, powder, tablet, capsule, solution, or emulsion, depends in part on the route by which it is administered. The therapeutically effective amount can be administered in a series of doses separated by appropriate time intervals, such as hours.
Pathogenic toxins which can be inhibited by the method of the present invention include, but are not limited to, toxins produced by a microorganism, such as bacteria, viruses, protozoa, fungi or parasites. Such toxins include bacterial toxins, such as those produced by Streptococcus, including Streptococcus pneumoniae, and Streptococcus pyogenes; Salmonella, including Salmonella enteritidis; Campylobacter, including Campylobacter jejuni; Escherichia coli; Clostridia, including Clostridium difficile and Clostridium botulinum; Staphylococcus, including Staphylococcus aureus; Shigella dysenteriae; Pseudomonas including Pseudomonas aeruginosa; Bordatella pertussis; Listeria monocytogenes; Vibrio cholerae; Yersinia enterocolitica; Legionella pneumophilia; and Bacillus anthracis. 
Of particular pathogenic importance are Escherichia coli, for example, E. coli strains 06:H-, 0157:H7,0143 and other clinical isolates, and Clostridium difficile. Enterohemorrhagic Esherichia coli (EHEC), such as 0157:H7, can cause a characteristic nonfebrile bloody diarrhea known as hemorrhagic colitis. EHEC produce high levels of one or both of two related cytotoxins which resemble a Shiga toxin in structure and function and are referred to as Shiga-like toxins (SLT I or SLT II). These Shiga toxins are believed to damage the intestinal mucosa, resulting in the manifestation of hemorrhagic colitis.
Clostridium difficile produce two major toxins, designated Toxin A and Toxin B, which cause damage to the cellular lining of the bowel wall. Toxin A causes fluid production and damage to the mucosa of the large bowel. Toxin B is a cytotoxin which causes abnormalities in tissue culture systems. This quality of Toxin B is used to diagnose the disease by detecting toxin in feces.
Also included are protozoal toxins, such as toxins produced by Entameoba histolytica, and Acanthameoba; and parasitic toxins.
The method of the invention can also be used to inhibit a viral toxin, such as a toxin produced by rotavirus, human immunodeficiency virus, influenza virus, polio virus, vesicular stomatitis virus, vaccinia virus, adenovirus, picomavirus, togaviruses (such as sindbis and semlikifores viruses), paramyxoviruses, papillomaviruses. Toxins which can be inhibited using the method of the invention include viroporin molecules produced by any of these viruses. A preferred toxin which can be inhibited using the method of the invention is the rotavirus NSP4 protein. Other toxins which can be inhibited include influenza M2 protein, HIV Vpu and gp41 proteins, picornavirus 3A protein, togavirus 6K protein, respiratory syncitial virus SH protein, coronavirus D3 protein and adenovirus E5 protein.
The method is useful for treating infections of various organs of the body, but is particularly useful for infections of the skin and gastrointestinal tract.
Polymers which are particularly suitable for the present method include polymers which can possess key characteristics of naturally occurring antigens, in particular, the ability to form amphipathic structures. The term xe2x80x9camphipathicxe2x80x9d, as used herein, describes a three-dimensional structure having discrete hydrophobic and hydrophilic regions. Thus, one portion of the structure interacts favorably with aqueous and other polar media, while another portion of the structure interacts favorably with non-polar media. An amphipathic polymer results from the presence of both hydrophilic and hydrophobic structural elements along the polymer backbone.
Polymers to be administered which have amino groups can be administered in the free base, amino form, or as a salt with a pharmaceutically acceptable acid. Such acids include hydrochloric acid, hydrobromic acid, citric acid, lactic acid, tartaric acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid, mucic acid, benzoic acid, glucoronic acid, oxalic acid, ascorbic acid, and acetylglycine. In either case, at physiological pH following administration, a plurality of amino groups will be protonated to become ammonium groups, and the polymer will carry an overall positive charge.
Polymers comprising quaternary ammonium groups will further comprise a pharmaceutically acceptable counter anion, such as an anion which is a conjugate base of one of the pharmaceutically acceptable acids discussed above. The number of counter anions associated with the polymer prior to administration is the number necessary to balance the positive charge on the polymer.
The polymer to be administered can be an addition polymer having a polymer backbone such as a polyacrylate, polyacrylamide, poly(allylalcohol), poly(vinylalcohol), poly(vinylamine), poly(allylamine), or poly(diallylamine) backbone. The polymer can have a uniform backbone if it is composed of monomers derived from a common polymerizable unit, such as acrylamide. If the polymer is a copolymer, it can also comprise a mixed backbone, a block copolymer backbone, a grafted backbone or an interpenetrating polymer backbone.
The polymers of use in the present method also include condensation polymers, wherein polymerization of monomers is accompanied by the release of a small molecule, such as a water molecule. Such polymers include, for example, polyesters and polyurethanes.
The polymers of use in the present method can be linear or crosslinked. The polymer can be crosslinked, for example, by the incorporation within the polymer of a multifunctional comonomer. Suitable multifunctional co-monomers include diacrylates, triacrylates and tetraacrylates, dimethacrylates, diacrylamides, diallylacrylamide, di(methacrylamides), triallylamine and tetraalylammonium ion. Specific examples include ethylene glycol diacrylate, propylene glycol diacrylate, butylene glycol diacrylate, ethylene glycol dimethacrylate, butylene glycol dimethacrylate, methylene bis(methacrylamide), ethylene bis(acrylamide), ethylene bis(methacrylamide), ethylidene bis(acrylamide), ethylidene bis(methacrylamide), pentaerythritol tetraacrylate, trimethylolpropane triacrylate, bisphenol A dimethacrylate, and bisphenol A diacrylate. Other suitable multifunctional monomers include polyvinylarenes, such as divinylbenzene. The amount of crosslinking agent is typically between about 1.0% and about 30% by weight relative to the weight of the polymer, preferably from about 5% to about 25% by weight.
The polymer can also be crosslinked by bridging units which link amino groups on adjacent polymer strands. Suitable bridging units include straight chain or branched, substituted or unsubstituted alkylene groups, diacylalkylene groups, diacylarene groups and alkylene bis(carbamoyl) groups. Examples of suitable bridging units include xe2x80x94(CH2)nxe2x80x94, wherein n is an integer from about 2 to about 20; xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94; xe2x80x94C(O)CH2CH2C(O)xe2x80x94; xe2x80x94CH2xe2x80x94CH(OH)xe2x80x94Oxe2x80x94(CH2)mxe2x80x94Oxe2x80x94CH(OH)xe2x80x94CH2xe2x80x94, wherein m is an integer from about 2 to about 4; xe2x80x94C(O)xe2x80x94(C6H2(COOH)2)xe2x80x94C(O)xe2x80x94 and xe2x80x94C(O)NH(CH2)pNHC(O)xe2x80x94, wherein p is an integer from about 2 to about 20.
Advantageously, crosslinking the polymers renders the polymers non-adsorbable and stable in the patient. A xe2x80x9cstablexe2x80x9d polymer composition, when administered in therapeutically effective amounts, does not dissolve or otherwise decompose to form potentially harmful byproducts, and remains substantially intact.
The polymer can be crosslinked, for example, by including a multifunctional co-monomer as the crosslinking agent in the reaction mixture. A multifunctional co-monomer can be incorporated into two or more growing polymer chains, thereby crosslinking the chains. Suitable multifunctional co-monomers include those discussed above. The amount of crosslinking agent added to the reaction mixture is, generally, between 1.0% and 30% by weight relative to the combined weight of the polymer and the crosslinking agent, and preferably from about 2.5% to about 25% by weight.
The multifunctional co-monomer can also take the form of a multifunctional diallylamine, such as a bis(diallylamino)alkane or a bis(diallylalkylammonio) alkane. Suitable monomers of this type include 1,10-bis(diallylmethylammonio)decane dibromide and 1,6-bis(diallylmethylammonio)hexane dibromide, each of which can be formed by the reaction of diallylmethylamine with the appropriate dibromoalkane.
In one embodiment, the polymer to be administered comprises a monomer, or repeat unit, of Formula I, 
wherein X is a covalent bond, a carbonyl group or a CH2 group, Y is an oxygen atom, an NH group or a CH2 group, Z is an spacer group, R is a hydrogen atom or a methyl or ethyl group; R1, R2 and R3 are each, independently, a hydrogen atom, a normal or branched, substituted or unsubstituted C1-C24-alkyl group, an aryl group or an arylalkyl group; Axe2x88x92 is a pharmaceutically acceptable anion, such as a conjugate base of a pharmaceutically acceptable acid; and m and n are each, independently, 0 or 1. Suitable alkyl substituents include halogen atoms, such as fluorine or chlorine atoms. A monomer of Formula 1 in which at least one of substituents R1, R2 and R3 is hydrogen can also exist in the free base, or amino, form in which a hydrogen substituent is absent and the nitrogen atom is electrically neutral.
In a preferred embodiment, one of R1-R3 is an ammonioalkyl group of the general formula 
wherein R4, R5 and R6 are each, independently, a hydrogen atom, a C1-C24 alkyl group, or an arylalkyl group; n is an integer from 2 to about 20, preferably from 3 to about 6; and Axe2x88x92 is a pharmaceutically acceptable anion. An ammonioalkyl group in which at least one of substituents R4, R5 and R6 is hydrogen can also exist in the free base, or amino, form in which a hydrogen substituent is absent and the nitrogen atom is electrically neutral. The group xe2x80x94N+(R4)(R5)(R6) can also be a heteroaryl group, such as a 5- or 6-membered heteroaryl group, such as a 1-pyridinio group. Preferably, at least one of R4, R5 and R6 is a C6-C24-alkyl group. Examples of suitable ammonioalkyl groups include, but are not limited to,
4-(dioctylmethylammonio)butyl; 3-(dodecyldimethylammonio)propyl;
3-(octyldimethylammonio) propyl; 3-(decyldimethylammonio)propyl;
5-(dodecyldimethylammonio)pentyl; 3-(cyclohexyldimethylammonio)propyl;
3-(decyldimethylammonio)-2-hydroxypropyl; 3-(tridecylammonio)propyl;
3-(docosyldimethylammonio)propyl; 4-(dodecyldimethylammonio)butyl;
3-(octadecyldimethylammonio)propyl; 3-(hexyldimethylammonio)propyl;
3-(methyldioctylammonio)propyl; 3-(didecylmethylammonio)propyl;
3-(heptyldimethylammonio)propyl; 3-(dimethylnonylammonio)propyl;
6-(dimethylundecylammonio)hexyl; 4-(heptyldimethylammonio)butyl;
3-(dimethylundecylammonio)propyl; 3-(tetradecyldimethylammonio)propyl
3-(1-pyridinium)propyl; in combination with a pharmaceutically acceptable anion.
When at least one of R1 to R6 is a hydrogen atom, the monomer can also exist in the free base, or amino form. The polymer comprising such a monomer can be administered in the free base form or in the protonated form, for example, as a salt of a pharmaceutically acceptable acid.
The spacer group Z is a component of the polymer side chain and connects the amino or ammonium group to the polymer backbone. The amino or ammonium group is, thus, a pendant group. The spacer group can be a normal or branched, saturated or unsaturated, substituted or unsubstituted alkylene group, such as a polymethylene group xe2x80x94(CH2)nxe2x80x94, wherein n is an integer from about 2 to about 24. Suitable examples include the propylene, hexylene and octylene groups. The alkylene group can also, optionally, be interrupted at one or more points by a heteroatom, such as an oxygen, nitrogen (e.g, NH) or sulfur atom. Examples include the oxaalkylene groups xe2x80x94(CH2)2O[(CH2)2O]n(CH2)2xe2x80x94, wherein n is an integer ranging from 0 to about 3.
Examples of monomers of Formula I having quaternary ammonium groups include:
N-(3-dimethylaminopropyl)acrylamide,
N-(3-trimethylammoniopropyl)acrylamide,
2-trimethylammonioethyl methacrylate,
2-trimethylammonioethyl acrylate,
N-(3-trimethylammoniopropyl)methacrylamide,
N-(6-trimethylammoniohexyl)acrylamide,
N-(3-trimethylammoniopropyl)acrylamide,
N-(4-trimethylammoniobutyl)allylamine,
N-(3-dimethyloctylammoniopropyl)allylamine,
N-(3-trimethylammoniopropyl)allylamine,
N-(3-(1-pyridinio)propyl)vinylamine and
N-(3-(1-pyridinio)propyl)allylamine.
Each of these monomers also includes a suitable counter anion. Examples of monomers of Formula I having an amino group include allylamine, vinylamine and N-(3-dimethylamino-propyl)acrylamide. Each of these monomers can also exist as a salt with a pharmaceutically acceptable acid.
In one embodiment, the repeat unit of Formula 1 is of the formula 
where R1, R2, R3 and Axe2x88x92 have the meanings given above for formula I. In a preferred embodiment, R1, R2 and R3 are each hydrogen. For example, the polymer can be polyallylamine which is protonated on at least a portion of the nitrogen atoms. In a preferred embodiment, the polymer is protonated polyallylamine which is cross-linked with a difunctional cross-linking agent as described above. For example, the protonated polyallylamine can be cross-linked with an epihalohydrin, such as epichlorohydrin. In a specific example, the polymer to be administered is poly(allylamine) hydrochloride cross-linked with 5 to 10% by weight epichlorohydrin.
In another embodiment, the polymer to be administered is characterized by a diallylamine repeat unit of Formula III: 
wherein R1 and R2 are each, independently, a hydrogen atom, a normal or branched, substituted or unsubstituted C1-C24-alkyl group, an aryl group or an arylalkyl group; and Axe2x88x92 is a pharmaceutically acceptable anion, such as a conjugate base of a pharmaceutically acceptable acid. Suitable alkyl substituents include halogen atoms, such as fluorine or chlorine atoms. A monomer of Formula III in which at least one of substituents R1 and R2 is hydrogen can also exist in the free base, or amino, form, in which a hydrogen substituent is absent and the nitrogen atom is electrically neutral. In a preferred embodiment, R1 is an ammonioalkyl group of Formula II, as described above.
In another embodiment, the polymer to be administered is a copolymer characterized by a first repeat unit of Formula III wherein both R1 and R2 are hydrogen and a second repeat unit of Formula III wherein R1 and R2 are each, independently, a C1-C24-alkyl group. Preferably, in the second repeat unit of Formula III, R1 is a methyl group and R2 is a linear or branched C1-C18 alkyl group. The polymer can be linear or cross-linked, as described above, and preferably includes from about 0.5 to about 20% by weight of a cross-linking agent, such as epichlorohydrin or one of the other difunctional cross-linking agents described above.
In another embodiment, the polymer to be administered is a poly(alkyleneimine) polymer comprising a monomer, or repeat unit, of Formula IV, 
wherein n is an integer from about 2 to about 10 and R7 and R8 are each, independently, a hydrogen atom, a normal or branched, substituted or unsubstituted C1-C24-alkyl group, an aryl group or an arylalkyl group, and Axe2x88x92 is a pharmaceutically acceptable anion. Suitable alkyl substituents include halogen atoms, such as fluorine or chlorine atoms. When one of R7 and R8 is a hydrogen atom, the polymer can be administered in the free base form or in the cationic form shown, as the salt of a pharmaceutically acceptable acid. A monomer of Formula IV in which at least one of substituents R7 and R8 is hydrogen can also exist in the free base, or amino, form, in which a hydrogen substituent is absent and the nitrogen atom is electrically neutral. In a preferred embodiment, the polymer to be administered is a poly(ethyleneimine) polymer, comprising a monomer of Formula IV wherein n is 2.
Preferably, R7 is an aminoalkyl group, or an ammonioalkyl group of Formula II, as described above. In one embodiment, the polymer comprises monomeric units of Formula II wherein R7 is an aminoalkyl group, or an ammonioalkyl group, as well as monomeric units wherein R7 and R1 are each hydrogen or R7 is hydrogen and R8 is absent. The fraction of monomeric units which include the aminoalkyl or ammonioalkyl group can be from about 5% to about 90% of the monomeric units of the polymer.
Suitable polymers comprising a monomer of Formula II include poly(decamethylenedimethylammonium-co-ethylenedimethylammonium) Xxe2x88x92, wherein Xxe2x88x92 is an anion, for example chloride or bromide; poly(ethyleneimine-co-N-decylethyleneimine-co-N-(trimethylammonio-propyl)ethyleneimine; poly(ethyleneimine-co-N-benzylethyleneimine).
The polymer to be administered can also be a copolymer comprising a monomer of Formula I, Formula III or Formula IV and further comprising a hydrophobic monomer. The hydrophobic monomer can comprise a side chain bearing a hydrophobic group, such as a straight chain or branched, substituted or unsubstituted C3-C24-alkyl group or a substituted or unsubstituted aryl group. Examples of suitable hydrophobic monomers include styrene, N-isopropylacrylamide, N-t-butylacrylamide, N-n-butylacrylamide, heptafluorobutyl acrylate, N-n-decylallylamine, N-n-decylacrylamide, pentafluorostyrene, n-butyl acrylate, t-butyl acrylate, n-decyl acrylate, N-t-butylmethacrylamide, n-decyl methacrylate, n-butyl methacrylate, n-hexyl methacrylate, N-n-hexylvinylamine, N-n-hexylallylamine, N-benzylallylamine, N-(cyclohexylmethyl)allylamine, N-(n-decyl)allylamine, N-hexylethyleneimine, N-(3-phenylpropyl)ethyleneimine, N-decylethyleneimine and N-benzylethyleneimine.
Examples of copolymers characterized by a monomer of Formula I and a hydrophobic monomer include poly(N-(3-dimethylaminopropyl)acrylamide-co-N-n-butylacrylamide) or salts thereof with pharmaceutically acceptable acids. Other examples of suitable copolymers include poly(2-trimethylammonioethylmethacrylate-co-styrene) chloride, poly(2-trimethylammonioethylmethacrylate-co-N-isopropylacrylamide) chloride, poly(2-trimethyl-ammonioethylmethacrylate-co-heptafluorobutylacryl) chloride, poly(3-trimethylammoniopropylmethacrylate-co-styrene) chloride, poly(3-trimethylammonium-propylmethacrylate-co-N-t-butylacrylamide) chloride, poly(3-trimethylammoniopropylmethacrylate-co-N-n-butylacrylamide) chloride, and poly(N-(3-trimethylammoniopropyl)allylamine-co-N-n-decylallylamine). Each of these ionic copolymers can also be employed with one or more counter anions other than chloride, for example, with a conjugate base of one or more pharmaceutically acceptable acids.
In a further embodiment, the polymer to be administered comprises a monomer of Formula I, Formula III or Formula IV, a hydrophobic monomer and a neutral hydrophilic monomer, such as acrylamide, methacrylamide, N-(2-hydroxyethyl) acrylamide or 2-hydroxyethylmethacrylate. Examples of polymers of this type include terpolymers of N-(3-trimethylammonium-propyl)methacrylamide/N-isopropyl-acrylamide/2-hydroxyethyl-methacrylate, N-(3-trimethylanmronium-propyl)methacrylamide/N-n-decylacrylamide/2-hydroxyethylmethacrylate, N-(3-trimethylammoniopropyl)methacrylamide/N-t-butylmethacrylamide/methacrylamide, N-(3-trimethylammonium-propyl)methacrylamide/n-decylacrylate/methacrylamide, 2-trimethylammonioethylmethacrylate/n-butyl-acrylate/acrylamide, 2-trimethylammonium-ethylmethacrylate/t-butylacrylate/acrylamide, 2-trimethylammonioethylmethacrylate/n-decyl-acrylate/acrylamide, 2-trimethylammonium-ethylmethacrylate/n-decylmethacrylate/methacrylamide, 2-trimethylammonioethylmethacrylate/N-t-butylmethacrylamide/methacrylamide and 2-trimethylammonioethylmethacrylate/N-n-butylmethacrylamide/methacrylamide.
In one embodiment, the polymer to be administered is a cross-linked polymer characterized by two or more monomers of Formula I and/or Formula II. Preferably, the cross-linked polymer is characterized by a first monomer having primary or secondary amino groups and a second monomer having tertiary-amino groups or quaternary ammonium groups. Suitable examples of the first monomer include, but are not limited to, allylamine, vinylamine, N-alkylallylamine, N-alkylvinylamine and diallylamine. Suitable examples of the second monomer include, but are not limited to, N-alkyldiallylamine, N,N-dialkylallylamonium Axe2x88x92, N,N-dialkylallylamine, N,N,N-trialkylallylammonium Axe2x88x92. In the foregoing monomers, Axe2x88x92 is a suitable anion and the alkyl groups are preferably linear or branched C1-C24-alkyl groups, more preferably C1-C4-alkyl groups, and most preferably methyl groups.
In one embodiment, the cross-linked polymer comprises two or more linear polymers which are cross-linked as a mixture by post-polymerization cross-linking. For example, the cross-linked polymer can be prepared by cross-linking two or more distinct linear polymers, that is, polymer strands having distinct chemical compositions. Preferably, the cross-linked polymer is prepared by cross-linking first and second linear polymers having distinct compositions. In one embodiment, the first linear polymer is characterized by a repeat unit having a primary amino group and/or a repeat unit having a secondary amino group and the second linear polymer is characterized by a repeat unit having a tertiary amino group and/or a repeat unit having a quaternary ammonium group. The second linear polymer can additionally include one or more repeat units having primary and/or secondary amino groups. Suitable examples for the first linear polymer include polyallylamine, polyvinylamine, poly(ethyleneimine), polydiallylamine, N-alkylallylamine, for example, N-methylallylamine, and N-alkylvinylamine, for example, N-methylvinylamine. The second linear polymer preferably includes repeat units having amino groups which will react readily with a difunctional cross-linking agent, as described above. For example the second linear polymer preferably includes repeat units having primary amino groups or repeat units having secondary amino groups in addition to the repeat units having tertiary amino groups or quaternary ammonium groups. Suitable examples of the second linear polymer include copolymers of N-alkyldiallylamine, N,N-dialkylallylamonium Axe2x88x92, N,N-dialkylallylamine, and N,N,N-trialkylallylammonium Axe2x88x92 with at least one additional monomer which can react with a cross-linking agent. For example, the second linear polymer can be poly(N-alkyldiallylamine-co-diallylamine); poly(N,N-dialkyldiallylamonium-co-allylamine) Axe2x88x92; poly(N,N-dialkylallylamine-co-allylamine); poly(N,N-dialkylallylamine-co-N-alkylallylamine); poly(N,N,N-trialkylallylammonium-co-allylamine) Axe2x88x92; poly(N,N,N-trialkylallylammonium-co-N-alkylallylamine) Axe2x88x92; poly(N,N-dialkylvinylamine-co-vinylamine); poly(N,N-dialkylvinylamine-co-N-alkylvinylamine); poly(N,N,N-trialkylvinylammonium-co-vinylamine) Axe2x88x92; and poly(N,N,N-trialkylvinylammonium-co-N-alkylvinylamine) Axe2x88x92.
The composition of the copolymers to be administered can vary substantially. The copolymer can comprise from about 95 mole percent to about 5 mole percent, preferably from about 20 mole percent to about 80 mole percent, of a monomer of Formula I. The copolymer can also comprise from about 95 mole percent to about 5 mole percent, preferably from about 20 mole percent to about 80 mole percent, of a hydrophobic monomer.
The polymers of use in the present method are preferably substantially nonbiodegradable and nonabsorbable. That is, the polymers do not substantially break down under physiological conditions into fragments which are absorbable by body tissues. The polymers preferably have a nonhydrolyzable backbone, which is substantially inert under conditions encountered in the target region of the body, such as the gastrointestinal tract.
Other examples of polymers which are of use in the present method are disclosed in U.S. patent application Ser. Nos. 08/482,969, 08/258,477, 08/258,431, 08/469,659 and 08/471,769, the contents of each of which are incorporated herein by reference.
The polymer to be administered will, preferably, be of a molecular weight which is suitable for the intended mode of administration and allows the polymer to reach and remain within the targeted region of the body for a period of time sufficient to interact with the toxin associated with the pathogen. For example, a method for treating an intestinal infection should utilize a polymer of sufficiently high molecular weight to resist absorption, partially or completely, from the gastrointestinal tract into other parts of the body. The polymers can have molecular weights ranging from about 500 Daltons to about 500,000 Daltons, preferably from about 2,000 Daltons to about 150,000 Daltons.
The polymers which are useful in the present method can be prepared by known methods. A first method includes the direct polymerization of a monomer, such as trimethylammonioethylacrylate chloride, or a set of two or more monomers, such as trimethylammonioethylacrylate chloride, N-n-butylacrylamide and acrylamide. This can be accomplished via standard methods of free radical, cationic or anionic polymerization which are well known in the art. Due to reactivity differences between two monomers, the composition of a copolymer produced in this way can differ from the composition of the starting mixture. This reactivity difference can also result in a non-random distribution of monomers along the polymer chain.
A second method proceeds via the intermediacy of an activated polymer comprising labile side chains which are readily substituted by a desired side chain. An example of a suitable activated polymer is the succinimide ester of polyacrylic acid, poly(N-acryloyloxysuccinimide) (also referred to hereinafter as xe2x80x9cpNASxe2x80x9d), which reacts with nucleophiles such as a primary amine to form a N-substituted polyacrylamide. Another suitable activated polymer is poly(para-nitrophenylacrylate); which reacts with amine nucleophiles in a similar fashion.
A copolymer having a polyacrylamide backbone comprising amide nitrogens bearing two different substituents can be prepared by treating pNAS with less than one equivalent (relative to N-acryloyloxysuccinimide monomer) of a first primary amine, producing a poly(N-substituted acrylamide-co-N-acryoyloxysuccinimide) copolymer. Remaining N-acryoyloxysuccinimide monomer can then be reacted with, for example, an excess of a second primary amine to produce a polyacrylamide copolymer having two different N-substituents. A variety of copolymer compositions can, thus, be obtained by treating the activated polymer with different proportions of two or more amines.
Polymers suitable for use in the present method can also be prepared by addition of a side chain to a preformed polymer. For example, poly(ethyleneimine), poly(allylamine) and poly(vinylamine) can each be alkylated at the amino nitrogen by one or more alkylating agents. For example, a fraction of the amino groups can be alkylated using an alkylating agent such as a normal or branched C3-C24-alkyl halide, such as n-decyl bromide, or an (X-alkyl)ammonium salt, wherein X represents a suitable leaving group, such as a halide, a tosylate or a mesylate group. These compounds can be prepared by the reaction of an appropriate dihaloalkane, such as a bromochloroalkane, with a tertiary amine. Suitable alkylating agents of this type include the following:
(4-bromobutyl)dioctylmethylammonium bromide;
(3-bromopropyl)dodecyldimethylammonium bromide;
(3-chloropropyl)dodecyldimethylammonium bromide;
(3-bromopropyl)octyldimethylammonium bromide;
(3-chloropropyl)octyldimethylammonium bromide;
(3-iodobutyl)dioctylmethylammonium bromide;
(2,3-epoxypropyl)decyldimethylammonium bromide;
(3-chloropropyl)decyldimethylammonium bromide;
(5-tosylpentyl)dodecyldimethylammonium bromide;
(6-bromohexyl)octyldimethylammonium bromide;
(12-bromododecyl)decyldimethylammonium bromide;
(3-bromopropyl)tridecylammonium bromide;
(3-bromopropyl)docosyldimethylammonium bromide;
(6-bromohexyl)docosyldimethylammonium bromide;
(4-chlorobutyl)dodecyldimethylammonium bromide;
(3-chloropropyl)octadecyldimethylammonium bromide;
(3-chloropropyl)hexyldimethylammonium bromide;
(3-chloropropyl)methyldioctylammonium bromide;
(3-chloropropyl)methyldidecylammonium bromide;
(3-chloropropyl)cyclohexyldimethylammonium bromide;
(3-bromopropyl)heptyldimethylammonium bromide;
(3-bromopropyl)dimethylnonylammonium bromide;
(6-bromohexyl)dimethylundecylammonium bromide;
(4-chlorobutyl)heptyldimethylammonium bromide;
(3-chloropropyl)dimethylundecylammonium bromide;
(3-chloropropyl)tetradecyldimethylammonium bromide; and
1-(3-chloropropyl)pyridinium bromide.
Each of the alkylating agents described above can also be prepared and used as a salt in combination with an anion other than bromide. For example, these and similar alkylating agents can be prepared and used as salts with a wide range of anions, including chloride, iodide, acetate, p-toluenesulfonate and methanesulfonate.
When substituents are added to the polymer by way of an alkylating agent as described above, the extent of alkylation can be determined by methods which are well known in the chemical arts. The increase in polymer mass due to alkylation provides a measure of the extent of alkylation. For example, in a reaction between poly(allylamine) and 1-bromohexane, a product/starting polymer mass ratio of about 3.9, 2.5 and 1.7 represent approximately 100%, 50% and 25% alkylation, respectively. The degree of alkylation can also be determined by elemental analysis of the product polymer. In this case, the carbon/nitrogen (C/N) mass ratio is a direct measure of the degree of alkylation. For example, the reaction of poly(allylamine) with 1-bromohexane yields a product with a higher C/N mass ratio than that of the starting polymer. Product C/N mass ratios of about 7.7, 5.1 and 3.9 represent, approximately, 100%, 50% and 25% alkylation, respectively.
The polymer can be crosslinked, for example, by including a multifunctional co-monomer as the crosslinking agent in the reaction mixture. A multifunctional co-monomer can be incorporated into two or more growing polymer chains, thereby crosslinking the chains. Suitable multifunctional co-monomers include those discussed above. The amount of crosslinking agent added to the reaction mixture is, generally, between 1.0% and 30% by weight relative to the combined weight of the polymer and the crosslinking agent, and preferably from about 2.5% to about 25% by weight.
The multifunctional co-monomer can also take the form of a multifunctional diallylamine, such as a bis(diallylamino)alkane or a bis(diallylalkylammonio) alkane. Suitable monomers of this type include 1,10-bis(diallylmethylammonio)decane dibromide and 1,6-bis(diallylmethylammonio)hexane dibromide, each of which can be formed by the reaction of diallylmethylamine with the appropriate dibromoalkane.
The polymers to be administered can also be crosslinked subsequent to polymerization by reacting the polymer with one or more crosslinking agents having two or more functional groups, such as electrophilic groups, which react with amine groups to form a covalent bond. Crosslinking in this case can occur, for example, via nucleophilic attack of the polymer amino groups on the electrophilic groups. This results in the formation of a bridging unit which links two or more amino nitrogen atoms from different polymer strands. Suitable crosslinking agents of this type include compounds having two or more groups selected from among epoxide, acyl-X and alkyl-X, wherein Xxe2x88x92 is a suitable leaving group, such as a halide, acylate, tosylate or mesylate group. Examples of such compounds include epichlorohydrin, succinyl dichloride, butanedioldiglycidyl ether, ethanedioldiglycidyl ether, pyromellitic dianhydride and dihaloalkanes. The crosslinking agent can also be an a,w-alkylene diisocyanate, for example OCN(CH2)pNCO, wherein p is an integer from about 2 to about 20. The polymer can be reacted with an amount of crosslinking agent equal to from about 0.5 to 40 mole percent relative to the amino groups within the polymer, depending upon the extent of crosslinking desired.
As discussed below in Example 56, several polymers described herein were tested for in vitro activity against Shiga toxins 1 and 2 and exhibited excellent toxin-inhibiting properties.